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NBR1 acts as an autophagy receptor for peroxisomes

Elizabeth Deosaran1, Kenneth B. Larsen2, Rong Hua1,3, Graeme Sargent1,3, Yuqing Wang1,3, Sarah Kim1,

Trond Lamark2, Miluska Jauregui1, Kelsey Law1,3, Jennifer Lippincott-Schwartz4, Andreas Brech5,Terje Johansen2 and Peter K. Kim1,3,*1Cell Biology Program, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada2Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway3Department of Biochemistry, University of Toronto, Toronto, ON M5G 1X8, Canada4Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA5Deptartment of Biochemistry, Institute for Cancer Research, Centre for Cancer Biomedicine, Oslo University Hospital, Oslo, Norway

*Author for correspondence (pkim@sickkids.ca)

Accepted 15 November 2012Journal of Cell Science 126, 939–952� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.114819

SummarySelective macro-autophagy is an intracellular process by which large cytoplasmic materials are selectively sequestered and degraded in

the lysosomes. Substrate selection is mediated by ubiquitylation and recruitment of ubiquitin-binding autophagic receptors such as p62,NBR1, NDP52 and Optineurin. Although it has been shown that these receptors act cooperatively to target some types of substrates tonascent autophagosomes, their precise roles are not well understood. We examined selective autophagic degradation of peroxisomes

(pexophagy), and found that NBR1 is necessary and sufficient for pexophagy. Mutagenesis studies of NBR1 showed that theamphipathic a-helical J domain, the ubiquitin-associated (UBA) domain, the LC3-interacting region and the coiled-coil domain arenecessary to mediate pexophagy. Strikingly, substrate selectivity is partly achieved by NBR1 itself by coincident binding of the J andUBA domains to peroxisomes. Although p62 is not required when NBR1 is in excess, its binding to NBR1 increases the efficiency of

NBR1-mediated pexophagy. Together, these results suggest that NBR1 is the specific autophagy receptor for pexophagy.

Key words: Selective autophagy, Pexophagy, Autophagy receptors, NBR1, p62, Peroxisomes

IntroductionPeroxisomes are critical metabolic organelles that are required

for oxidation of fatty acids, and reduction of hydrogen peroxide

produced during lipid oxidation. They are required for synthesis

of essential cellular components such as plasmalogens,

isoprenoids and lysine. Unlike other metabolic organelles,

peroxisome numbers are highly regulated, varying in numbers

depending on cellular needs (Till et al., 2012). This plasticity is

most readily observed in the liver of rodents. Upon activation of

the nuclear receptor peroxisome proliferator-activated receptor-

alpha (PPARa), peroxisome proliferation occurs by growth and

division of pre-existing peroxisomes (Platta and Erdmann, 2007).

However, upon removing the PPARa ligand, peroxisome

numbers are quickly reduced to basal levels. This reduction is

mediated by macro-autophagy, a catabolic mechanism of

delivering large cytosolic material to lysosomes for degradation

(Iwata et al., 2006). Little is known about the precise mechanism

of macro-autophagy of peroxisomes, or macro-pexophagy

(hereafter referred to as pexophagy). However, it is known that

the inability to maintain peroxisome numbers is linked to various

neurodegenerative and developmental disorders such as X-linked

adrenoleukodystrophy, and Krabbe disease (Ribeiro et al., 2012;

Singh et al., 2009). In particular, in some leukodystrophies, the

loss of peroxisomes during neuroinflammation is thought to

exacerbate the cellular inflammation, eventually leading to cell

death (Kassmann and Nave, 2008).

Selective macro-autophagy (hereafter selective autophagy) is a

catabolic cellular process by which large cytoplasmic materials

are selectively sequestered and degraded in lysosomes. In the

mammalian system, peroxisomes along with other cytosolic

components such as protein aggregates, damaged mitochondria,

ER, ribosomes, membrane remnants, midbody rings, intracellular

bacteria, bacteriocidal precursor and viral capsid proteins are

substrates of selective autophagy (reviewed by Johansen and

Lamark, 2011; Kirkin et al., 2009b; Komatsu and Ichimura,

2010; Kraft et al., 2010). The mechanism of selective autophagy

is not well understood. However, recent studies of various

substrates are coalescing into a common theme in the mechanism

of targeting selective substrates to autophagosomes. In this

mechanism the substrate is activated for degradation by

accumulation of ubiquitin (Ub) on the cytosolic side of the

substrate followed by recruitment of ubiquitin-binding autophagy

receptor(s) (Johansen and Lamark, 2011). The autophagy

receptors play an essential role in linking the substrate to

nascent autophagosomes by binding to both the ubiquitin on the

substrate and the autophagy factor LC3 on the autophagosomes.

At present four Ub-binding mammalian autophagy receptors

have been identified: p62, NBR1, NDP52 and optineurin

(Bjørkøy et al., 2005; Kirkin et al., 2009a; Pankiv et al., 2007;

Thurston et al., 2009; Wild et al., 2011). Recent studies suggest

that depending on the substrate, these Ub-binding receptor

proteins can either act cooperatively or independently. For

example, both p62 and NBR1 have been reported to act

cooperatively to target polyubiquitylated aggregates to

autophagosomes (Kirkin et al., 2009a). However, Salmonella

targeting to autophagosomes does not require NBR1, but instead

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requires both p62 and NDP52 (Cemma et al., 2011; Zheng et al.,2009). These two autophagy receptors do not act cooperativelybut appear to be recruited to Salmonella independently of each

other. Similar results were observed for Listeria. However, forShigella the recruitment of all three proteins was shown to beinterdependent on each other (Mostowy et al., 2011).

The role of the various Ub-binding receptors in selective

autophagy of organelles is not well understood. Several groupshave reported that p62 is involved in mitophagy (the autophagicdegradation of mitochondria); however, it is not clear whetherp62 is required for targeting of ubiquitylated mitochondria to

autophagosomes or serves a different functional role (Ding et al.,2010; Geisler et al., 2010; Narendra et al., 2010; Okatsu et al.,2010). Recently, we showed that p62 is involved in selective

degradation of peroxisomes when peroxisomes were labeled withubiquitin (Kim et al., 2008). Depleting cells of p62 using siRNAresulted in a decrease in peroxisome turnover, thus causing an

increase in peroxisome numbers. However, the role of NBR1 inpexophagy is not known.

Here, we tested the hypothesis that autophagy receptors confersubstrate selectivity in autophagy. For these studies we used

peroxisomes as substrates for selective autophagy and examinedthe role of two autophagy receptors, NBR1 and p62. Peroxisomeswere selected as the substrate to examine the role of these tworeceptors for two reasons. First, peroxisome numbers can be

readily quantified, allowing for monitoring changes in theirnumbers both statically and dynamically. Second, except for ourprevious report implicating p62 in the clearance of peroxisomes

(Kim et al., 2008), little is known about the role of otherautophagy receptors, like NBR1, in mammalian pexophagy.Here, we characterize the roles of both NBR1 and p62 in

targeting of peroxisomes to autophagosomes for theirdegradation. Based on our findings we present a mechanism forsubstrate specificity in selective autophagy. NBR1 uses amechanism similar to ‘coincidence detection’, where the

combination of the ubiquitin-binding UBA domain and thephospholipid-binding J domain functions as an effective‘coincidence detector’ (Carlton and Cullen, 2005), allowing

NBR1 to target peroxisomes for autophagic degradation.

ResultsNBR1 is required for the turnover of peroxisomes

Recently, NBR1 and p62 were shown to act together toselectively target polyubiquitylated protein aggregates to

autophagosomes for degradation (Kirkin et al., 2009a). p62 wasalso shown to be involved in pexophagy (Kim et al., 2008).However, it is not known whether NBR1 is required forperoxisome degradation, and if so, whether it works

cooperatively with p62. In order to address this question, wedepleted HeLa cells of NBR1 using siRNA and examinedwhether its loss affected the endogenous level of peroxisomes. To

quantify the change in peroxisome numbers, the totalfluorescence intensity from a peroxisomal protein, catalase, wasquantified by indirect immunofluorescence (Huang et al., 2011;

Kim et al., 2008). To ensure that only cells with knockdown ofp62 and/or NBR1 were quantified, they were co-immunostainedfor p62 and/or NBR1 expression.

As shown previously (Kim et al., 2008), knocking down p62

expression resulted in an increase in catalase levels relative to thecells treated with non-targeting siRNA, suggesting that p62depletion inhibited the endogenous turnover of peroxisomes

(Fig. 1A,B). Similarly, in cells where NBR1 was depleted, an

increase in catalase levels was also observed, suggesting that

NBR1 is also involved in the endogenous turnover of

peroxisomes (Fig. 1A,B). The increase in peroxisomal catalase

levels in p62 and NBR1 knockdown cells was confirmed by

western blot analysis, which showed elevated catalase levels in

cells where either NBR1 or p62 was depleted (Fig. 1C). We also

examined the autophagy receptor NDP52, which was previously

shown to be involved in autophagy of the bacterial pathogen

Salmonella typhimurium (Thurston et al., 2009). When NDP52

expression was knocked down using siRNA, the endogenous

level of peroxisomes was not affected (supplementary material

Fig. S1). Hence, NDP52 is not required for basal peroxisome

turnover. Together, these experiments suggest that NBR1 and

Fig. 1. NBR1 is required for pexophagy. (A) HeLa cells were transfected

with non-targeting siRNA (siCtrl), siRNA against p62, NBR1, or against both

p62 and NBR1, as indicated, over two consecutive days. Two days later, the

cells were fixed and immunostained for catalase, NBR1 and p62. Scale bars:

10 mm. (B) The normalized mean fluorescence intensity of catalase levels of

at least 100 cells in A. The average (n53) for each condition was normalized

against the control non-targeting siRNA (siCtrl). Only cells showing a

decrease in NBR1 and/or p62 expression were quantified, with the exception

of the control experiment. Error bars indicate s.d. ***P,0.001, **P50.001–

0.01. (C) Total cell lysates were prepared from siRNA-treated cells (as in A)

and immunoblotted with catalase, NBR1, p62 and actin antibodies. In the

NBR1 blot, two bands are present. The arrow indicates the NBR1 band and

the asterisk indicates a cross-reacting band.

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p62, but not NDP52, are required for basal turnover ofperoxisomes.

NBR1 and p62 act in the same pathway

Interestingly, the increase in catalase levels in NBR1 knockdowncells was significantly higher than those observed in p62knockdown cells (Fig. 1B,C). A possible explanation for thisobservation is that p62 and NBR1 mediate independent but

parallel peroxisome degradation pathways, like that found withNDP52 and p62 in S. typhimurium (Cemma et al., 2011).Conversely, both may be involved in the same pathway but at

different stages. To determine whether p62 and NBR1 act in twodistinct parallel pathways, we knocked down the expression ofboth p62 and NBR1 simultaneously and quantified peroxisomal

catalase levels (Fig. 1B). If NBR1 and p62 are involved indifferent but parallel pathways, depletion of both proteins shouldresult in an additive increase in peroxisome levels. If NBR1 and

p62 are involved in the same pathway but at different steps, thenno additive increase should be observed when both proteins aredepleted. As shown in Fig. 1B, when both NBR1 and p62 wereknocked down, the increase in peroxisome levels was not

additive but remained similar to NBR1-only knockdownconditions. This result was further verified by western blot ofendogenous catalase levels that showed no further increase in

catalase upon knockdown of both p62 and NBR1 compared toNBR1 alone (Fig. 1C). Thus, NBR1 and p62 act in the samepathway.

Exogenously expressed NBR1 induces peroxisomeclustering

If p62 and NBR1 are involved in the same pathway, they mayfunction cooperatively to target peroxisomes to autophagosomes,similarly to polyubiquitylated protein aggregates. If this is the

case, then the differential inhibition of pexophagy observedbetween NBR1 and p62 knockdown conditions (Fig. 1B)suggests that NBR1 may play the initiating or prominent role

in mediating pexophagy. This hypothesis is supported by theobservation that knockdown of NBR1 resulted in a similarincrease in catalase levels compared to cells where autophagy

was inhibited by knockdown of the autophagic factor ATG12(Fig. 1B,C; supplementary material Fig S2). A possibleexplanation for these results is that NBR1 may be recruited toperoxisomes to activate the targeting of peroxisomes to

autophagosomes before p62. If this model is true, thenoverexpression of NBR1 should upregulate pexophagy byincreasing the amount of free NBR1 that can bind to

peroxisomes.

We tested this hypothesis by first asking whether exogenouslyexpressed NBR1 colocalized with endogenous peroxisomes.

Peroxisomes were identified using antibodies againstendogenous catalase or PMP70 (Fig. 2A,F). When weexpressed a Cherry–NBR1 chimera protein, we observed that

peroxisomes clustered around NBR1-positive punctate structures(Fig. 2B,C). Peroxisome clustering was not observed in cellsexpressing Cherry–p62 (Fig. 2D,E). In order to illustrate the

differences in peroxisome binding between the two autophagyreceptors, we quantified the Manders’ colocalization coefficientof Cherry signal overlapping with catalase/PMP70 (MCherry) for

both autophagy receptors. For these quantifications, only cellswith similar expression levels (as determined by the total Cherryfluorescence in each cell) were quantified. In comparison to

Cherry–p62, Cherry–NBR1 showed a significantly highercolocalization coefficient with endogenous catalase and PMP70

(Fig. 2G). Similar NBR1/peroxisome clusters were observed inother cell lines, such as COS7, HEK293 and wild-type mouseembryonic fibroblasts (MEFs), showing that the observedphenomenon was not cell type specific (data not shown).

To determine the nature of these NBR1/peroxisome clusters,we examined HeLa cells expressing NBR1 by correlative light–electron microscopy (CLEM). As seen in the representative

images (Fig. 3A–D), we found two populations of NBR1-positivestructures. One group consisted of clusters of single membranevesicular-like structures with a diameter of 40–70 nm (Fig. 3D,

arrow). The second population clearly shows these vesicular-likestructures sequestered within a double-membrane structureresembling typical autophagosomes (Fig. 3D, arrowhead). Theidentity of these NBR1 clusters was further verified by immuno-

EM on cells transfected with GFP-NBR1 (Fig. 3E). To establishthe relationship between these NBR1 vesicle structures andperoxisomes, we transfected HeLa cells with Cherry–NBR1 and

the peroxisomal marker GFP–PMP34 (Fig. 3F–H). At highmagnification we observed many peroxisome-like vesicles,often localized at the periphery of the aggregates (Fig. 3I,K).

These results confirm the clustering of peroxisomes with NBR1observed by confocal fluorescence microscopy.

NBR1 promotes targeting of peroxisomes to lysosomes

To determine whether the peroxisome/NBR1 clusters lead totargeting of peroxisomes to lysosomes we looked forcolocalization with the lysosomal marker Lamp1

(supplementary material Fig. S3). To prevent degradation ofperoxisomes and NBR1 within lysosomes, and allow theirvisualization, cells were treated with the lysosomal protease

inhibitors E-64 and leupeptin. The majority of the NBR1 punctatestructures colocalized with Lamp1 (supplementary material Fig.S3A). This colocalization was abolished upon treating the cells

with the autophagy inhibitor 3-methyladenine (3-MA)(supplementary material Fig. S3B), suggesting that thecolocalization of NBR1 with lysosomes was mediated byautophagy. We also examined whether peroxisome/NBR1

clusters also colocalized with lysosomes. When Cherry–NBR1/Lamp1–GFP-expressing cells were probed for endogenouscatalase, the majority of peroxisomes were found clustered with

NBR1 within lysosomes (supplementary material Fig. S3C–E).To determine the extent of this clustering we quantified thenumber of the NBR1/peroxisome clusters that colocalized with

the lysosomal marker Lamp1. In these cells 85613% of theseclusters were found inside lysosomes. However, there was asmall population of cells (,10%) that had large NBR1aggregates with peroxisomes clustered around them but not

localized inside lysosomes. The other 5% on average did notcolocalize with peroxisomes. Time-lapse imaging of cellsexpressing Cherry–NBR1 and the peroxisomal marker PMP34-

GFP verified that peroxisomes were targeted to lysosomes fordegradation when NBR1 was overexpressed. As the expression ofNBR1 increased, peroxisomes clustered around NBR1, and

eventually disappeared (supplementary material Movie 1). Nextwe asked whether NBR1/peroxisome clusters localize withautophagosomes. In cells expressing GFP–NBR1 along with

the autophagosome marker Cherry–LC3 we found that NBR1/peroxisome clusters colocalized with Cherry–LC3-positivestructures (supplementary material Fig. S3F,G). Together, these

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results strongly suggest that exogenously expressing NBR1

causes an increased targeting of peroxisomes to lysosomes byautophagy.

In order to visualize and quantify pexophagy activity due toelevated NBR1 levels, we employed a pH-sensitive live-cell

assay hereafter referred to as the ‘RG-lysosome assay’ (Pankiv

et al., 2007). In this assay a tandem chimera of mCherry andEGFP fused to the peroxisome membrane targeting sequence of

PEX26 (GFP–Cherry–PEX26TM) is expressed in HeLa cells.

Since the fluorescence of mCherry is more acid resistant thanEGFP, peroxisomes targeted to lysosomes maintain their red

fluorescent signal while the GFP fluorescence gets quenched.

Thus, GFP–Cherry–PEX26TM localized inside lysosomesexhibits red fluorescence only, while cytoplasmic protein

appears yellow due to the combination of red and green

fluorescence (supplementary material Fig. S4). Time lapseimaging of cells expressing Cer–NBR1 and GFP–Cherry–

PEX26TM over 2 days revealed an NBR1-mediated gradual

movement of peroxisomes into lysosomes, followed bydisappearance of peroxisomes (supplementary material Movie

2). The disappearance of peroxisomes was preceded by an

increase in ‘Red’ peroxisomes (which appear slightly purple due

to overlap with the blue signal from Cer–NBR1). Unlike GFP,Cerulean has a low pKa (4.7), thus it maintains its fluorescence

signal within the low pH environment of lysosomes (Rizzo et al.,

2004).

In order to assess the ability of exogenously expressed NBR1

to induce pexophagy we quantified pexophagy using the RG-lysosome assay in cells coexpressing GFP–Cherry–PEX26TM

with Cer–NBR1, Cer–p62 or Cerulean (Cer). We considered a

cell to show increased pexophagy if 20% or more of its totalCherry pixels were ‘red-only’ (supplementary material Fig. S4).

A pixel was considered to be ‘red-only’ if the fluorescent signal

from Cherry was at least three times higher than the GFP signal(see Materials and Methods). There was a significant increase in

the number of cells with elevated pexophagy upon expressing

Cer–NBR1 compared to Cer–p62 or Cerulean alone (Fig. 4A,B).Interestingly, no significant difference was observed between

cells expressing Cer–p62 compared to Cerulean alone (Fig. 4B).

To demonstrate that the increase in the red-only signal was due topexophagy, we treated the cells with 3-MA and found that it

prevented the accumulation of red-only signal (Fig. 4A,B).

Fig. 2. Exogenously expressed NBR1 promotes peroxisome

clustering and targeting to lysosomes. (A) HeLa cells transfected

with Cherry-N1 and immunostained for either catalase or PMP70 as

indicated. (B,C) A representative image of HeLa cells transiently

transfected with Cherry-NBR1 and immunostained for endogenous

catalase (B), or PMP70 (C). (D,E) Cells were transiently transfected

with Cherry-p62 and immunostained for catalase (D) or PMP70 (E).

Also shown on the right in B–E are enlarged images of the boxed

regions in the merge panels. (F) Mock-transfected HeLa cells

immunostained for either catalase or PMP70. (G) Bar graph of the

percentage of Cherry–NBR1 or Cherry–p62 colocalized with either

endogenous catalase or PMP70, as indicated. Percentage

colocalization was determined by calculating Manders’ colocalization

coefficient. At least 50 cells were quantified for each set of three

independent experiments. Error bars indicate s.d. All images are single

confocal images. Scale bars: 10 mm.

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To further verify that exogenously expressing NBR1 leads to

pexophagy we repeated these experiments in ATG5 knockout

MEFs, an autophagy deficient cell line. To quantify changes in

peroxisome numbers, we immunostained the MEFs for

endogenous catalase and determined the peroxisome density

(number of peroxisomes per cell volume). There was no

significant change in peroxisome numbers in Atg52/2 MEFs

when Cherry–NBR1 was exogenously expressed, whereas a

significant loss of peroxisomes was observed in wild-type (Atg5+/+)

MEFs compared to non-transfected cells (Fig. 4C,D). These results

were further verified by immunoblot analysis for changes in

peroxisomal proteins. We found that both catalase and PMP70

levels decreased in the wild-type cells but not in the autophagy

deficient cell line (Fig. 4E). Therefore, these results suggest that

NBR1 can promote the activation of pexophagy. Interestingly, we

found that there is no difference in the percentage of NBR1 clustered

with peroxisomes between the two cell lines (Fig. 4F), yet there is a

significant decrease in peroxisomes (Fig. 4E). These results may

indicate that the clustering of NBR1 with peroxisomes precedes

targeting to autophagosomes/lysosomes.

Fig. 3. Ultrastructural characterization of NBR1

aggregates by correlative light/electron

microscopy. (A) HeLa cells were transfected with

GFP-NBR1 for 24 hours and CLEM analysis was

performed. (B) Spatial alignment of the electron

micrograph with the confocal image. (C) Higher

magnification image with direct overlay of the

electron micrograph and the fluorescent image. (D) At

higher magnification the GFP–NBR1-positive

structures could be identified as aggregate-like

structures (arrows in D), which were found either in a

cytosolic, non-membrane-bound form or clearly

sequestered within double-membrane-surrounded

vesicles, resembling typical autophagosomes

(arrowhead in D). (E) The GFP–NBR1 clusters were

also apparent by immuno-EM using antibodies against

GFP. The relationship between NBR1 aggregates and

peroxisomes was further investigated in HeLa cells

transfected with Cherry-NBR1 and the peroxisomal

marker GFP–PMP34. (F) Confocal microscopy

revealed very frequent colocalization of peroxisomes

with Cherry–NBR1-positive structures (arrows in F).

(G,H) Further CLEM analysis of the sample revealed

similar cytosolic aggregates as in cells transfected

with only GFP-NBR1. (I,K) At higher magnification

we observed many peroxisome-like vesicles (enlarged

in the inset in I, arrows in K), which often seemed to

be localized at the periphery of the aggregates. Scale

bars as indicated.

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NBR1 can promote pexophagy independently of p62

The activation of pexophagy by elevated NBR1 levels suggests

that the amount of NBR1 in the cell may partly regulate

pexophagy. The fact that NBR1 upregulated pexophagy at

endogenous p62 levels suggests that p62 may not be strictly

required for pexophagy during elevated NBR1 levels. Hence, we

asked whether NBR1 could target peroxisomes to lysosomes

independently of p62. To ascertain the requirement for p62 in

NBR1-dependent pexophagy, we repeated the NBR1 RG-

lysosome assay in cells where p62 expression was knocked

down. In p62 siRNA-treated cells, Cer–NBR1-expressing cells

still showed only a slight decrease in pexophagy compared to

Cer–NBR1-expressing cells treated with non-targeting siRNA

(Fig. 5A). However, these cells showed a significant increase in

‘red-only’ signal compared to control (Cer) or Cer–p62-

expressing cells (Fig. 5A). Again there was no significant

difference between cells expressing the siRNA resistant Cer–

p62 and Cerulean alone, in both control siRNA and p62

knockdown conditions. These results suggest that NBR1 is

sufficient to target peroxisomes to lysosomes for degradation in

the absence of p62.

p62 increases the efficiency of NBR1-mediated pexophagy

The observations that p62 was not necessary in the presence of

elevated NBR1 expression (Fig. 5A) but was required at

endogenous NBR1 levels (Fig. 1) suggest that p62 may play a

Fig. 4. Increased pexophagy in response to exogenous expression of NBR1. (A) Representative images of cells cotransfected with GFP-Cherry-PEX26TM and

Cerulean (Cer), Cer-NBR1 or Cer-p62. Also shown is a representative cell treated with 3-MA. (B) Quantification of the percentage of cells in A that had increased

pexophagy, using the RG-lysosome assay (see Materials and Methods for definition of increased pexophagy). (C) The number of catalase-positive punctate

structures per cell volume of cells in D were quantified and normalized to their respective untransfected cells. (D) Atg5+/+ and Atg52/2 MEFs were transfected

with Cherry-NBR1 and immunostained for endogenous catalase. (E) Immunoblot analysis of Atg5+/+ and Atg52/2 MEFs, either mock transfected or transfected

with Cherry-NBR1. (F) A graph of the percentage of Cherry–NBR1 that colocalized with peroxisomes (catalase). The average of at least three independent

experiments with standard deviations, determined from at least 50 cells from each experiment, is shown. **P50.001–0.01, ns P.0.05. Scale bars: 10 mm.

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secondary role in the targeting of peroxisomes to lysosomes. To

test this hypothesis, we compared the efficiency of pexophagy in

cells expressing a mutant of NBR1, which is deficient in p62-

binding (NBR1 D50R) to wild-type NBR1. NBR1 interacts with

p62 via its PB1 domain and a single residue mutation within the

PB1 domain (D50R) prevents this interaction (Kirkin et al.,

2009a). To directly compare the D50R mutant with wild-type

NBR1, siRNA-resistant (SR) versions of NBR1 constructs were

expressed in cells where endogenous NBR1 was knocked down

using siRNA. Under these conditions the NBR1 D50R mutant

was less efficient in inducing pexophagy than wild-type NBR1

(Fig. 5B). The decrease in efficiency was not due to the

instability of the mutant protein, since the expression of this

and subsequently discussed mutant NBR1 proteins was checked

by western blot analysis (supplementary material Fig. S5B).

To verify that p62 does interact with NBR1 during pexophagy,

we examined the cellular localization of endogenous p62 in cells

expressing Cherry–NBR1. We found that endogenous p62

colocalized with the NBR1/peroxisome clusters (Fig. 5C).

Interestingly, close examination of these NBR1/p62/peroxisome

clusters showed that p62 did not completely overlap with NBR1,

but also formed punctate structures juxtaposed to the NBR1/

peroxisome cluster structures (Fig. 5C).

Next we examined whether p62 was required for NBR1/

peroxisome cluster formation. We found that even upon knocking

down the endogenous p62, NBR1 was able to induce clustering

of peroxisomes (Fig. 5D). Combined with the observation that

elevated p62 expression alone did not cause peroxisome

degradation (Fig. 4B; Fig. 5A), but was required for pexophagy

at endogenous levels of NBR1 (Fig. 1), the above results suggest

that p62 is not involved in the clustering and may not be required

for NBR1 interaction with peroxisomes. Its role in pexophagy

may be to increase the efficiency of NBR1-mediated targeting of

peroxisomes to autophagosomes.

Homo-oligomerization of NBR1 is required for NBR1-

dependent pexophagy

The homo-oligomerization of p62 is required for its targeting to

nascent autophagosomes (Itakura and Mizushima, 2011). p62

homo-oligomerizes through the self-interaction of its PB1

domain (Lamark et al., 2003; Wilson et al., 2003). Although

NBR1 also possesses a PB1 domain (through which it interacts

with p62), it instead forms homo-oligomers via its coiled-coil

domain (CC1) (Kirkin et al., 2009a). It is not known, however,

whether self-oligomerization is required for targeting its cargo to

autophagosomes. In order to address this question, we expressed

Fig. 5. NBR1 promotes pexophagy independently of p62, but p62

increases efficiency of NBR1-mediated pexophagy. (A) Bar graph of

the percentage of cells showing increased pexophagy as determined by

the RG-lysosome assay. Cells treated with either control siRNA or

siRNA against p62 were transfected with Cer, Cer-NBR1, or siRNA-

resistant Cer-p62 plasmids. The percentage of cells with increased

pexophagy was determined by counting at least 50 cells. (B) Bar graph

of the percentage of cells showing increased pexophagy in NBR1

knockdown cells expressing either siRNA resistant Cer–NBR1 or Cer–

NBR1(D50R). For A and B the means 6 s.d. were calculated from

three independent experiments. **P50.001–0.01. (C) Cells expressing

Cherry–NBR1were fixed and immunostained for endogenous catalase

and p62. (D) p62-siRNA-treated cells were transfected with Cherry–

NBR1 and immunostained for p62 and catalase as in C. The boxed

regions in C and D are shown enlarged in the bottom row in each

panel. Scale bars: 10 mm.

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the NBR1 construct, NBR1 DCC1, where the coiled-coil domain

was deleted. This construct is defective in homo-oligomerization

(Kirkin et al., 2009a). Although this mutant can still cause

peroxisome clustering, clustering was less efficient in the absence

of endogenous wild-type NBR1 (Fig. 6A,B). Additionally, the

RG-lysosome assay indicated that it targeted peroxisomes to

lysosomes much less efficiently than wild-type NBR1 (Fig. 6C).

In fact, there was no significant difference between NBR1 DCC1

compared to the control vector (Cer). Therefore, these results

suggest that homo-oligomerization of NBR1 is required for

efficient targeting of peroxisomes to autophagosomes.

LIR motifs of NBR1 are required for pexophagy

One characteristic of autophagy receptors is their ability to

interface between the substrate and autophagosomes. The latter is

accomplished by the LC3-interacting region (LIR), which is

required for autophagy receptors to bind the autophagy factor

LC3. It has been previously shown that NBR1 mediates targeting

of polyubiquitylated aggregates to autophagosomes and that

NBR1 contains two LIRs (LIR1 and LIR2) (Kirkin et al., 2009a).

To determine whether the LIRs of NBR1 are required for

pexophagy we constructed an NBR1 mutant where LIR1 is

mutated and LIR2 deleted. This construct, called NBR1(LIRmut),

formed peroxisome clusters at similar levels as wild-type NBR1

(Fig. 6D). However, its pexophagy activity was significantly

decreased compared to the wild-type NBR1 (Fig. 6E), suggesting

that the LIR motifs are required for pexophagy and not for

NBR1/peroxisome clustering.

The J and UBA domains of NBR1 are required for

pexophagy

Previously we showed that an increase in ubiquitylated proteins

on peroxisomes resulted in their targeting to autophagosomes for

degradation (Kim et al., 2008). This suggests that the ubiquitin

binding domain of NBR1, the UBA domain, may be required for

binding to peroxisomes. Recently, the JUBA domain (hereafter

for simplicity referred to as the J domain) – a novel, membrane-

interacting, amphipathic a-helix region preceding the UBA

domain – was found to be necessary for binding to endosomes

(Mardakheh et al., 2010). Hence, we wanted to test if the J

domain is also required for peroxisome binding.

First, we determined whether these domains were required for

clustering of peroxisomes with NBR1. When both the J and UBA

domains were deleted (GFP–NBR1 DJ-DUBA), clustering was

Fig. 6. Homo-oligomerization of NBR1 and LIR is required for

pexophagy. (A) HeLa cells treated with either control siRNA or

siRNA against NBR1 were transfected with an siRNA-resistant Cer-

NBR1(DCC1) and immunostained for endogenous catalase. Shown

are maximum projections of Z-stack images of 1.4 mm slices. Scale

bars: 10 mm. (B) Quantification of percentage of cells showing

colocalization of NBR1 with peroxisomes. Data are from three

independent experiments (n.50). The percentage of NBR1

colocalized with catalase was determined using the Manders’

M(Cherry) coefficient from a single confocal image. The two

averages were significantly different (P,0.01). (C) RG-lysosome

assay for pexophagy in NBR1 knockdown cells expressing either

NBR1SR, Cer–NBR1(DCC1)SR or Cerulean. The percentage shown is

the average of three independent experiments (n.50). ***The Cer–

NBR1(DCC1)SR-expressing cells were significantly different from

NBR1SR cells (P,0.001). (D) Quantification of colocalization of

Cherry–NBR1 and Cherry–NBR1(LIRmut) with peroxisomes as in B.

(E) RG-lysosome assay for pexophagy for cells expressing either

NBR1 or NBR1(LIRmut). ***The Cer–NBR1(LIRmut)-expressing

cells were significantly different from NBR1SR cells (P,0.001).

Values are means 6 s.d.

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not observed (Fig. 7A,E). Similarly, deletion of the J domain

(NBR1 DJ) also abrogated formation of peroxisome/NBR1

clusters (Fig. 7B,E). However, when only the UBA was deletedand the J domain retained (NBR1 DUBA), clustering occurred

(Fig. 7C). The extent of clustering was quantified by calculating

the percentage of NBR1 that colocalized with peroxisomes, using

the Manders’ colocalization coefficient. The colocalization of

NBR1 DUBA with peroxisomes was similar to that of wild-typeNBR1 (Fig. 7E).

Interestingly, the NBR1 DUBA mutant localized to bothpunctate and tubular structures that resembled mitochondria

(Fig. 7C). Upon further investigation, this mutant was indeed

found to colocalize with the mitochondrial marker mito-RFP in

HeLa cells (Fig. 7D) and COS7 cells (data not shown). However,

NBR1, NBR1 DJ and NBR1 DJ-DUBA were not found tocolocalize with mitochondria (data not shown). We also examined

the colocalization of the NBR1 DUBA mutant with other

organelles. A comparison of colocalization coefficients of NBR1

DUBA versus the wild-type NBR1 show that there is increased

colocalization of NBR1 DUBA with the ER, lysosomes, the Golgi

apparatus and mitochondria (supplementary material Fig. S6). p62

does not have any domain homologous to the J domain. When we

fused the J domain to p62 with its own UBA domain deleted, we

also found that p62-JN-DUBAN localized to mitochondria

(supplementary material Fig. S7D–F). The wild-type p62 or

another variant where the J domain is located N-terminal to the

UBA domain of p62 does not localize to mitochondria

(supplementary material Fig. S7A–C). Therefore, these

observations suggest that the J domain is capable of binding

membrane bilayers other than peroxisomes in the absence of the

UBA domain. Hence, the UBA domain may play a regulatory role

in the targeting of NBR1 to specific membranes.

Fig. 7. Both the J domain and the UBA domain of NBR1 are

required to induce pexophagy. HeLa cells were transfected

with plasmids encoding (A) GFP–NBR1 DJDUBA; (B) Cer–

NBR1 DJ; or (C) Cer–NBR1 DUBA. The cells were fixed and

immunostained for endogenous catalase. The boxed area is

enlarged in the images labeled ‘inset’. (D) GFP–NBR1 DUBA-

expressing cells were cotransfected with mito-RFP. The boxed

area is enlarged in the images below. (E) Colocalization of

Cherry-tagged NBR1 or p62 with endogenous catalase.

Manders’ thresholded colocalization coefficients were

determined for at least 50 cells for each condition. Error bars

represent standard error of the mean; **P,0.01. Averages of

three independent experiments are shown. (F) Quantification of

the RG-lysosome assay for pexophagy in cells expressing the

autophagy receptor proteins and mutants as indicated. HeLa

cells were transfected with plasmids expressing both the

autophagy receptor and GFP–Cherry–PEX26 and live-cell

images were acquired. The averages from at least three

independent experiments are shown. At least 50 cells were

quantified for each set of experiments. **P,0.001 compared to

p62. Scale bars: 10 mm.

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To determine whether the various NBR1 deletion mutants can

promote pexophagy, we coexpressed them with GFP–Cherry–

PEX26TM and subjected them to the RG-lysosome assay. The

three mutants NBR1 DUBA, NBR1 DJ and NBR1 DJ-DUBA did

not show any significant increase in pexophagy compared to

control (Fig. 7F). Only the wild-type NBR1, containing both the

J and UBA domains, resulted in a significant increase in

pexophagy. Therefore, these results show that although the J

domain is required for peroxisome binding, both the J domainand UBA domains are required for targeting peroxisomes to

autophagosomes for lysosomal degradation.

The J domain of NBR1 enables p62 to mediate pexophagy

To further test the role of the UBA and J domains in pexophagy,

we examined whether they can confer pexophagy activity on p62.

p62 has similar domain architecture to NBR1; it contains all the

domains required to induce pexophagy, except for the

amphipathic J domain preceding the UBA domain (Fig. 8A).

Like NBR1, p62 forms homo-oligomers, possesses a UBA

domain and possesses an LIR domain. Yet p62 cannot induce

pexophagy upon overexpression in cells (Fig. 4B). Therefore, we

asked whether the J-domain of NBR1 confers its specificity for

peroxisomes. To test this hypothesis, we constructed a number of

domain-swap mutants of p62 where its UBA domain was

replaced with the J and/or UBA domains of NBR1 (Fig. 8A).

We also inserted the J domain of NBR1 immediately upstream of

the UBA domain in p62 (p62-JN-UBAp). All chimeras containing

the J domain exhibited clustering with peroxisomes (Fig. 8B), but

when they were subjected to the RG-lysosome assay, only the

p62 chimeras containing both the J domain and a UBA domain

(of either NBR1 or p62) were able to cause an increase in

pexophagy (Fig. 8C; supplementary material Fig. S8). Notably,

the UBA domains of NBR1 and p62 were interchangeable and

both promoted pexophagy, provided that the J domain was also

present. The constructs containing only the J domain or UBA

domain of NBR1 did not induce pexophagy above the level found

for wild-type p62 and Cer control. Together with the analysis of

NBR1 mutants these data suggest that both the J and UBA

domains are required to promote selective degradation of

peroxisomes.

Inhibiting PEX5 recruitment to peroxisomes preventspexophagy

Next we asked how NBR1 might be recruited to peroxisomes.

The importance of the UBA domain for selective binding of

NBR1 to peroxisomes suggests that NBR1 is likely binding to a

ubiquitylated protein on peroxisomes. One possible candidate is

PEX5, a cytosolic receptor for peroxisomal matrix proteins.

Peroxisome matrix proteins are imported into peroxisomes by the

formation of a transient pore that consists of at least two proteins,

PEX14 and PEX5. PEX14 is the membrane receptor that recruits

the matrix protein receptor PEX5 to the peroxisomal membrane,

and together they form a transient pore (Meinecke et al., 2010). It

is thought that this transient pore is disassembled by

ubiquitylation of PEX5 (Platta et al., 2007). We wanted to test

if NBR1 is recruited to peroxisomes upon binding to

ubiquitylated PEX5. To test this hypothesis we prevented

PEX5 recruitment to peroxisomes by knocking down the

expression of PEX14. Using quantitative PCR we confirmed

that there was a 74% decrease in PEX14 mRNA upon treating

HeLa cells with siRNA against PEX14 (Fig. 8D). Furthermore

we observed that knocking down PEX14 disrupted the import of

Fig. 8. The J domain of NBR1 is sufficient to confer

peroxisome specificity on p62 and PEX14 knockdown

decreases NBR1-induced pexophagy. (A) Schematic

diagram of NBR1 and p62 domain mutants. CC, coiled

coil domain; LIR, LC3-interacting domain.

(B,C) Quantification of clustering (B) and the RG-

lysosome assay (C) of HeLa cells transfected with

Cerulean or Cerulean-tagged autophagy receptor

proteins and mutants, as indicated. The means 6 s.d.

from three independent experiments are shown. At least

50 cells were quantified for each set of experiments,

n53. *P,0.001 compared to Cerulean-expressing cells.

(D) Quantitative PCR of PEX14 mRNA levels in HeLa

cells treated with control siRNA and PEX14 siRNA.

(E) Quantification of pexophagy by the RG-lysosome

assay in HeLa cells with PEX14 knockdown and NBR1

overexpression. HeLa cells were transfected with

siRNAs and Cerulean-NBR1, as indicated, and GFP–

Cherry–PEX26, and live cell images were acquired. The

means 6 s.d. from at least three independent

experiments are shown. At least 50 cells were quantified

for each set of experiments. *P,0.01; **P,0.001.

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a PEX5 cargo, catalase, into peroxisomes, (supplementarymaterial Fig. S9A, punctate versus cytosolic), suggesting that

PEX14 knockdown prevents PEX5 recruitment to peroxisomes.

To determine whether NBR1 required ubiquitylated PEX5 totarget to peroxisomes, we exogenously expressed Cherry–NBR1

in PEX14 siRNA knockdown cells. While Cherry–NBR1clustered with PMP70-positive structures in PEX14-containingcells, in PEX14 depleted cells we found that Cherry–NBR1 did

not colocalize with PMP70 positive structures, suggesting that itdid not bind to peroxisomes (supplementary material Fig. S9B–E). When we examined these cells for changes in pexophagy

upon NBR1 expression, we found that there was a significantdecrease in pexophagy compared to control siRNA-treated cells.Yet, pexophagy was still higher than control (Fig. 8E). This maybe the result of the incomplete knockdown of PEX14 (Fig. 8D;

supplementary material Fig. S9), or it is equally likely that NBR1is also binding to another ubiquitylated peroxisomal membraneprotein. Nevertheless, these experiments suggest that the

ubiquitylated PEX5 may be one component that recruits NBR1to peroxisomes.

DiscussionPeroxisome numbers are highly regulated. Pexophagy rapidly

removes superfluous or damaged peroxisomes (Till et al., 2012).Peroxisomes are also turned over in response to various cellularsignals, such as starvation conditions. During amino aciddeprivation peroxisomes are believed to be selectively degraded

while mitochondria are protected from autophagic degradation(Gomes et al., 2011; Hara-Kuge and Fujiki, 2008; Rambold et al.,2011). The ubiquitin binding autophagy receptors are involved in

selective targeting of organelles to autophagosomes fordegradation. Here we demonstrate that substrate selectivity maybe partly conferred by the autophagy receptors themselves. Using

peroxisomes as substrates for selective autophagy we showed thatNBR1, and not p62, is necessary and sufficient for the turnover ofendogenous peroxisomes.

We report that the J and UBA domains of NBR1 are requiredfor its specificity for peroxisomes. These domains conferspecificity by mediating the selective binding of NBR1 to

peroxisomes. An essential role for the J domain is supported bythe observation that mutants lacking the J domain (NBR1 DJ andNBR1 DJDUBA) did not colocalize with peroxisomes (Fig. 7).However, based on our results we hypothesize that the UBA

domain is required for the specific recruitment of NBR1 to theperoxisomal membrane. NBR1 without the UBA domain (NBR1DUBA) formed peroxisome/NBR1 clusters, but also exhibited

increased colocalization with other organelle markers, such as themitochondria, which was not observed with full length NBR1(supplementary material Fig. S6). Similar non-selective binding

to various bilayers via the J domain was also observed with thep62-JN-DUBA construct, where the UBA domain of p62 wasreplaced with the J domain of NBR1 (Fig. 8; supplementary

material Fig. S7). Adding a UBA domain from either p62 orNBR1 to the J domain (p62-JN-UBAP/N) prevented thenonspecific binding to mitochondria (supplementary materialFig. S7). These results suggest that the UBA domain may act as a

regulator of NBR1 binding to peroxisomes, since the J domain inthe absence of the UBA domain appears to bind non-specificallyto any lipid bilayer.

There is a precedent for this type of membrane bindingmechanism. A number of PI3P binding proteins, such as EEA1

and Vps27, target and bind to PI3P rich membranes via a two-step binding mechanism (reviewed in Kutateladze, 2006). Both

EEA1 and Vps27 possess the PI3P binding domain, FYVE, and amembrane interaction loop (MIL). These proteins are initiallytargeted to a specific membrane by interaction of the FYVEdomain with PI3P, resulting in a conformational change exposing

the membrane interaction loop domain to insert into the lipidbilayer to anchor the protein to the target organelle. In fact,phosphoinositide-binding proteins are known to act as detectors

of coincident localization signals to allow localization to specificmembranes and membrane domains (reviewed in Carlton andCullen, 2005). So, NBR1 may act as a coincidence detector

requiring both the coincident biding of the UBA domain and the Jdomain to peroxisomes to effectively target peroxisomes forautophagic degradation. We favor this mechanism as it explainsthe non-specific binding of the UBA deletion mutant (NBR1

DUBA) and the inability of the J domain deletion mutant (NBR1DJ) to bind to peroxisomes.

Interestingly, although NBR1 DUBA was able to bind

peroxisomes, it was not able to increase the pexophagy levels.Instead, an increase in pexophagy required both the J and UBAdomains. The inability of the J domain alone to induce

pexophagy may in part be explained by the fact that it binds tomore different organelles (substrates) than the wild type, whichappears to bind mainly to peroxisomes. The non-selective

binding of this mutant to a number of subcellular membranesmay result in an increase in autophagy substrates, causing aneffective decrease in peroxisome degradation.

The need for a UBA domain on NBR1 to induce pexophagy

further supports our previous report showing that increasingubiquitin moieties on the cytosolic face of peroxisomes inducedpexophagy (Kim et al., 2008). However, the question remains as

to which peroxisomal protein(s) is ubiquitylated to inducepexophagy. Here we demonstrate that one possible peroxisomalprotein target is PEX5, a component of the peroxisomal transient

pore complex that is required for the import of peroxisomalmatrix proteins. PEX5 is likely the most frequently ubiquitylatedprotein on the peroxisomal membrane, as it must be removed todisassemble the transient pore and be recycled back to the cytosol

to import other peroxisomal matrix proteins (Platta et al., 2007;Meinecke et al., 2010). We postulate that the overexpression ofNBR1 increases the rate of NBR1 interaction with the

ubiquitylated PEX5. This stabilizes the J-domain-mediatedbinding of NBR1 to the bilayer, resulting in the accumulationof NBR1 on the peroxisomal membrane. Pexophagy is likely

induced when there is sufficient accumulation of NBR1 onperoxisomes. In other words, in order to target peroxisomes toautophagosomes, a ‘critical mass,’ or number of autophagy

receptors on peroxisomes is likely required to target theseorganelles to nascent autophagosomes.

We found that knocking down p62 expression results in anincrease in peroxisome numbers, but not to the same levels as

observed for NBR1 or ATG12 knockdown (Fig. 1). Although wefind that p62 is not required for pexophagy in an NBR1overexpression system, it does increase efficiency. Thus, based

on our critical mass hypothesis, NBR1 on peroxisomes likelyrecruits p62 in order to increase the number of autophagy receptorson the peroxisome surface and achieve the ‘critical mass’ required

for efficient pexophagy. This is further supported by theobservation that although both NBR1 and p62 can formindependent punctate structures (Kirkin et al., 2009a), they

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colocalize with each other on the substrate (Fig. 6C). Interestingly,the colocalization of p62 with NBR1 was only partial as there were

distinct NBR1 and p62 structures juxtaposed to each other aroundperoxisomes. These structures may be analogous to themicrodomains formed by the autophagy receptors p62 and

NDP52 around Salmonella inside autophagosomes (Cemma et al.,2011; Mostowy et al., 2011; Zheng et al., 2009). However, thenature of these microdomains is not known.

Our finding that NBR1 is a receptor for pexophagy inmammalian cells suggests a mechanistic difference between the

yeast and mammalian systems. Recently, Atg30 has beendescribed to be the autophagy receptor for peroxisome turnoverin Pichia pastoris (Farre et al., 2008). This protein is found

localized on the peroxisomal membrane via its interaction withtwo peroxisomal membrane proteins, Pex14p and Pex3p. Unlikemammalian autophagy receptors it does not directly interact with

Atg8, the yeast LC3 homologue, but instead interacts with Atg11and Atg17 in order to target peroxisomes to autophagosomes.This interaction with the Atg11–Atg17 complex requires the

phosphorylation of Atg30. Furthermore, Atg30 does not have anobvious ubiquitin binding domain, suggesting that its targeting toperoxisomes is not ubiquitin dependent. However, a pexophagyreceptor, Atg36, that binds to Pex3 on peroxisomes and to Atg8

and the adaptor Atg11 was recently discovered in Saccharomyces

cerevisiae (Motley et al., 2012). Apart from the lack of ubiquitinin the targeting role, this is more similar to the situation with

NBR1 in mammalian pexophagy described here.

Materials and MethodsPlasmids

Cherry-NBR1, GFP-NBR1(DCC1), Cherry-NBR1(D50R), Cherry-p62, Lamp1-GFP, Lamp1-Cer, ss-GFP-KDEL, mito-RFP, mito-GFP, GFP-LC3 and PMP34-GFP were described previously (Kim et al., 2008; Kirkin et al., 2009a; Mitra et al.,2009). All other plasmids were constructed using standard protocols or Gatewayrecombination cloning, and were sequenced for confirmation (The Centre forApplied Genomics, Toronto, ON, Canada, and Sequencing Core Facility,University of Tromsø, Norway). Primers were produced by Sigma Genosys(Oakville, ON, Canada) and Invitrogen. Cloning strategies and primer sequencesused are available upon request.

The GFP-NBR1(DJDUBA) construct contains the first 875 codons of the NBR1gene, while GFP-NBR1(JDUBA) has the first 919 codons of NBR1 withinClontech pEGFP-C1. NBR1(D50R)(DJ) group of plasmids were constructed fromNBR1(D50R) plasmid but the base pairs pertaining to residues 877–902 weredeleted. For simplicity, these constructs, although D50R mutants, are referred to asNBR1(DJ) in the text and figures. SiRNA-resistant (SR) alleles of wild-type NBR1and the NBR1(DCC1) and NBR1(D50R) mutants were constructed by introducinga silent mutation at L839 by site-directed mutagenesis. A schematic representationof the constructs of Cer-p62-JNUBAN, p62-UBAN, p62-JN-DUBA and p62-JN-UBAp are shown in Fig. 8A.

GFP-Cherry-PEX26TM was constructed in several steps. First, the PEX26 ORFfrom pSPORT-PEX26 (Invitrogen) was cloned into the EcoRI and SalI sites ofpEGFP-C1, and then it was truncated by PCR. Finally, the mCherry gene wasinserted into the BglII and BamHI sites of the vector to generate GFP-Cherry-PEX26TM.

Reagents and antibodies

Leupeptin (Bioshop, or Enzo Life Sciences) was used at 0.5 mM and E-64 (EnzoLife Sciences) at 2 mM. 3-methyladenine (3-MA; Sigma-Aldrich) was dissolveddirectly in media at 10 mM. siRNA targeting NBR1 (59-GGAGUGGAUUUA-CCAGUUAUU-39), p62 (59-GCAUUGAAGUUGAUAUCGAUTT-39), ATG12(59-GUGGGCAGUAGAGCGAACATT-39), and non-targeting controls (59-UU-CUCCGAACGUGUCACGUTT-39 or 59-UAAGGCUAUGAAGAGAUACTT-39)were produced by GenePharma (Shanghai, China). The NDP52 siRNA (59-UUCAGUUGAAGCAGCUCUGUCUCCC-39) and the rabbit polyclonal anti-NDP52 was a gift from Dr John Brumell.

Primary antibodies used in this study were rabbit polyclonal anti-catalase (EMDBiosciences, Calbiochem, Darmstadt, Germany), mouse monoclonal anti-p62 (BDBiosciences), rabbit polyclonal anti-NBR1 (Kirkin et al., 2009a), mouse monoclonalanti-NBR1 (Abnova, Taipei City, Taiwan), rabbit polyclonal anti-GFP (T. Johansen)and mouse anti-actin (Sigma-Aldrich). All fluorescent anti-mouse and anti-rabbit

Alexa Fluor 488, 568 and 633 secondary antibodies were from Invitrogen.HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG secondaryantibodies were from Cedarlane. 10 nm Protein A gold (CMC, Utrecht, TheNetherlands).

Cell culture

HeLa and MEF cells were grown in DMEM supplemented with 10% fetal bovineserum (FBS; Invitrogen). For live-cell imaging, cells were grown, manipulated andimaged in LabTek chamber slides (Nalgene Nunc, Rochester, NY). Forimmunofluorescence, cells were grown either in LabTek chamber slides or onno. 1 glass coverslips. Plasmids were transfected using Fugene-HD (Roche) orLipofectamine-2000 (Invitrogen) or TransIT LT1 (Mirus Bio). siRNAs weretransfected using Lipofectamine-2000 according to the manufacturer’sinstructions. For simultaneous siRNA knockdown and plasmid overexpression,siRNA alone was transfected on the first day, and siRNA and plasmids weretransfected simultaneously on the second day, using Lipofectamine-2000. For theRG-lysosome assay, cells were treated with leupeptin and E-64 beginning on theday after plasmid transfection, to prevent degradation of protein inside lysosomesand allow detection of Cherry signal. When specified, cells were treated with 3-MA, beginning on the day after plasmid transfection. Cells were imaged 2 and/or 3days after plasmid transfection. Prior to live imaging, the medium was changed toCO2-independent medium (Invitrogen) containing 10% FBS, and leupeptin, E-64,and 3-MA, as needed.

Microscopy

Laser-scanning confocal microscopy was performed on a Zeiss LSM710 with a636/1.4 Plan-Apochromat oil objective or on a Leica TCS SP5 with a 636/1.2 NAwater immersion objective. When required, images were acquired in Z-sections of0.5 to 1.0 mM thickness. Live cell imaging was performed at 37 C in CO2-independent medium containing 10% FBS, and leupeptin, E-64 and 3-MA, asspecified. For immunofluorescence, cells were fixed using 3.7% paraformaldehyde(Electron Microscopy Sciences) in PBS for 15 minutes and permeabilized using0.1% Triton X-100 (Fisher Scientific) in PBS for 15 minutes, followed byincubation with the appropriate primary and secondary antibodies for 2 hours (orovernight at 4 C), and 1 hour, respectively. For all NBR1/peroxisome clusteringexperiments, the same pre-set microscopy acquisition setting was always used(laser power, master gain, etc.) to ensure that images from experiments performedon different days could be directly compared.

Correlative light and electron microscopy

Correlative light and electron microscopy (CLEM) was performed on HeLa cellsgrown on gridded coverslips (EMS, Hatfield, PA, USA) and transfected with GFP-NBR1 alone or double transfected with GFP-PMP34 and Cherry-NBR1 for24 hours as described earlier. Cells were fixed in 4% formaldehyde, 0.2%glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, embedded in Moviol andobserved on a confocal microscope (LSM 710, Carl Zeiss MicroImaging, Inc.)with 636 or 206 objectives, using the appropriate channels and differentialinterference contrast (DIC) microscopy. We used maximum projections from Z-stacks (seven slices, 4–6 mm total height) taken at 636magnification for the finaloverlay images. The localization of cells of interest on the gridded coverslip wasrecorded using DIC microscopy. After detaching the coverslips in phosphate buffer(1–2 hours at room temperature) they were fixed in 2% glutaraldehyde, followedby 2% OsO4 and 1.5% KFeCN in the same buffer. Coverslips were then stained enbloc with 4% uranyl acetate for 1 hour, dehydrated in graded ethanol series andembedded with Epon filled gelatin capsules placed on top of the coverslip. Thecoverslips were removed after polymerization with 48% hydrofluoric acid (Merck,Germany) and the blocks were then trimmed down to the regions previouslyidentified on the confocal microscope and now imprinted on the Epon block.Sections of 150–200 nm thickness were then cut parallel to the substratum on aLeica Ultracut and post-stained with lead citrate (2 minutes, Fig. 8B–D) or notstained (Fig. 8F–I). Electron micrographs were taken at indicated magnificationsand overlaid with the confocal images using Adobe Photoshop. Importantly, wewould like to point out that the confocal images represent total fluorescence fromthe whole cell volume, whereas electron micrographs only represent 150–200 nmthick sections, thereby explaining the fluorescent signals outside apparent cellborders and above the nuclei. Cells for immuno-EM were prepared as describedpreviously (Bjørkøy et al., 2005) and immunolabeled with antibodies against GFPfollowed by 10 nm Protein A gold.

Image analysis

Images were analyzed using Volocity software (Perkin Elmer). For quantificationof peroxisome intensity by catalase staining, all conditions were treatedsimultaneously using the same antibody conditions. All mean intensity wasnormalized against the control siRNA performed on the same day. Furthermore, tocapture the mean intensity from the catalase staining within the whole cell, imageswere acquired with an open pinhole. At least 100 cells were quantified, normalizedagainst the control and averaged per experiment. For quantification of peroxisome

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density in MEFs, the number of catalase-stained structures was divided by thevolume of each cell, and the averages were normalized to the Atg5+/+ untransfectedcontrols. At least 50 cells were analyzed for each condition.

For quantification of the RG-lysosome assay, all images were acquired usingsettings calibrated such that the GFP and mCherry fluorescence intensities (IG/R)were approximately equal (i.e. IGFP/ImCherry51). We found that this acquisitionsetting gave equivalent readings even on different days. Therefore a presetacquisition setting was used for all RG-lysosome assays. A cell was considered tobe undergoing increased pexophagy if the number of ‘red-only’ pixels were greaterthan 20% of the total ‘red’ pixels. To calculate the red-only pixels, all the pixelswith Cherry signal above a predetermined threshold were first determined, andboth the GFP (green) and Cherry (red) signals were collected for each of thesepixels. A pixel was considered red-only if the Cherry fluorescent signal was atleast three times higher than the GFP fluorescent signal. A cell was considered tohave an increase in pexophagy if at least 20% of the Cherry containing pixels werered-only (supplementary material Fig. 4). The percentage of cells with 20% ormore red-only pixels was reported.

Manders’ thresholded colocalization coefficients were determined for at least 50cells for each condition using ImageJ (NIH, Bethesda) and the JaCoP plugin (Bolteand Cordelieres, 2006). Cells above a pre-determined expression level, asdetermined by the fluorescence signal from NBR1 expression levels, were used.The threshold was set to exclude structures not corresponding to NBR1/p62 punctaor peroxisomes. The percentages reported correspond to the amount of mCherry–positive pixels that are also catalase-positive. Error bars represent standard error ofthe mean. For statistical significance a two-tailed t-test was performed usingMicrosoft Excel 2010.

Western blots

Cells were lysed with 100 mM Tris pH 9 containing 1% SDS (Bio-Rad) and Haltprotease inhibitor cocktail (Thermo Scientific), and the lysate was heated at 100 Cwith vortexing for 20 minutes, then centrifuged at 13,000 g for 20 minutes. Theprotein concentration in the supernatant was determined by BCA assay (EMDNovagen), equivalent sample amounts were subjected to SDS-PAGE, and proteinwas transferred to 0.45 mm BioTrace PVDF membrane (Pall) and probed with theappropriate primary and HRP-conjugated secondary antibodies following standardprotocols. Blots were developed using Luminata Crescendo (Millipore), ECL Plusor ECL Advance reagents (GE Healthcare) and detected on blue film (AGFA) orHyperfilm (GE Healthcare).

AcknowledgementsWe are indebted to Dr John Brumell for reagents and helpfuldiscussion pertaining to the project and the BioImaging core facilityat the Institute of Medical Biology, University of Tromsø for the useof instrumentation and expert assistance.

FundingThis work was funded by an operating grant from the CanadianInstitutes of Health Research [grant number MOP-111164 toP.K.K.]; an infrastructure grant from Canada Foundation forInnovation [grant number 23584 to P.K.K.]; the Ontario ResearchFund [grant number 23584 to P.K.K.]; the Functional Genomics andthe Biology and Biomedicine (FRIBIO) programs of the NorwegianResearch Council [grant number 196898 to T.J.]; and the NorwegianCancer Society [grant number 71043-PR-2006-0320 to T.J.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.114819/-/DC1

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